CN103421713B - For plant lactobacillus and the subtilis of the probiotic solid fermentation of feed additive field - Google Patents
For plant lactobacillus and the subtilis of the probiotic solid fermentation of feed additive field Download PDFInfo
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- CN103421713B CN103421713B CN201310322177.9A CN201310322177A CN103421713B CN 103421713 B CN103421713 B CN 103421713B CN 201310322177 A CN201310322177 A CN 201310322177A CN 103421713 B CN103421713 B CN 103421713B
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Abstract
A kind of pollute little, easy to operate, be conducive to gastrointestinal disturbances absorb the plant lactobacillus for fodder additives probiotic solid fermentation and subtilis, it is characterized in that: described subtilis (Bacillus? subtilis) HM-66 and plant lactobacillus (Lactobacillus? plantarum) HM-20 is probiotic bacterium, being preserved in China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation center, does is preserving number respectively CGMCC? No.6733 and CGMCC? No.6744.Preservation date is: on October 29th, 2012, the invention also discloses its application method.
Description
Technical field
The invention belongs to fodder additives probiotic solid fermentation field, especially pollute little, easy to operate, to be conducive to gastrointestinal disturbances the absorption plant lactobacillus for fodder additives probiotic solid fermentation and a subtilis, be applied to the aspects such as animal feedstuff additive.
Background technology
At present, probiotic composition many employings probiotic bacterium directly with liquid cow's milk and soya-bean milk fermentation, obtain functional yogurt milk or soybean milk yoghurt, all the other then adopt liquid state fermentation, and directly collection probiotic bacterium thalline, is made into probiotic bacterium pulvis, tablet or capsule.Adopt liquid state fermentation can produce a large amount of waste water, environmental pollution is comparatively large,
Aftertreatment is more difficult, machinery equipment complex operation.
Summary of the invention
The object of the invention is to adopt subtilis HM-66 and plant lactobacillus HM-20 to carry out mixed solid fermentation dregs of beans, provides a kind of and pollutes little, easy to operate, to be conducive to gastrointestinal disturbances the absorption subtilis for fodder additives probiotic solid fermentation and plant lactobacillus.
The present invention is achieved through the following technical solutions.
For plant lactobacillus and the subtilis of fodder additives neck probiotic solid fermentation, it is characterized in that: described subtilis (Bacillussubtilis) HM-66 and plant lactobacillus (Lactobacillusplantarum) HM-20 is probiotic bacterium, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is respectively CGMCCNo.6733 and CGMCCNo.6744, preservation date is: on October 29th, 2012, preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
For the plant lactobacillus of fodder additives probiotic solid fermentation and the application of subtilis, it is characterized in that: the cultivation of described subtilis HM-66 and plant lactobacillus HM-20 and solid state fermentation thereof are cultivated, obtains the solid state fermentation soybean products of probiotic bacterium.
For the plant lactobacillus of fodder additives probiotic solid fermentation and the using method of subtilis, it is characterized in that comprising the following steps: to adopt subtilis HM-66 and plant lactobacillus HM-20 to carry out mixed solid fermentation, described subtilis HM-66 and plant lactobacillus HM-20 seed culture medium are: raffinose 2%, dregs of beans 1%, add water to 100%, pH7.0-7.2.Solid-state fermentation culture medium: 58% dregs of beans, the sugar of 2%, moisture content 40%.
Described subtilis HM-66 seed culture condition, be positioned on shaking table, rotating speed is 150r/min, and temperature is 37 DEG C, cultivates 20h.The seed culture condition of described plant lactobacillus HM-20, is placed in constant incubator quiescent culture, culture temperature 37 DEG C, and incubation time is 20h.
Described solid state fermentation culture condition is: subtilis HM-66 and plant lactobacillus HM-20 inoculum size are respectively 1.0% and 4.0%.
At the fermentation initial stage, subtilis HM-66 and plant lactobacillus HM-20 mixed fermentation, pH is all on a declining curve, and pH minimum value is 5.44, and in the later stage of fermentation, its pH is all in rising trend, and after fermentation 48h, its pH is 6.60.
After subtilis HM-66 and plant lactobacillus HM-20 mixed fermentation 48h, its free ammonical nitrogen content is: 529.1umol/g.
Subtilis HM-66 and plant lactobacillus HM-20 mixed fermentation, after fermentation 48h, little peptide (<1000Da) content adds 81.3%, and large peptide (>10000Da) decreases 77.2%.
Advantage of the present invention and positively effect:
1. in the present invention, adopt subtilis HM-66 and plant lactobacillus HM-20 to carry out mixed solid fermentation dregs of beans.
2. the present invention adopts solid state fermentation production cost low, and the fund of investment is less, and convenient in downstream processing, pollute little, device structure is simple, easy to operate;
3. adopt plant lactobacillus HM-20 to ferment, the tart flavour such as lactic acid and acetic acid small-molecule substance can be produced, these small-molecule substances give improvement and the variation of the organoleptic features such as the distinctive sour fragrance of product, color and luster and mouthfeel, make bean pulp fermentation product more easily by human consumer is accepted;
4. adopt plant lactobacillus HM-20 fermented bean dregs, the protein in dregs of beans can be made to be broken down into little peptide and total free aminoacids, be beneficial to digesting and assimilating of stomach, the probiotic solid fermentation technology of feed additive field can be widely used in.
Accompanying drawing explanation
Fig. 1 is that plant lactobacillus HM-20 inoculum size is on the schematic diagram of the impact of viable count;
Fig. 2 is the schematic diagram of subtilis HM-66 inoculum size on the impact of plant lactobacillus HM-20 viable count;
Fig. 3 be the present invention when solid state fermentation, the time is on the schematic diagram of the impact of plant lactobacillus HM-20 viable count;
Fig. 4 is subtilis HM-66 and plant lactobacillus HM-20 when carrying out mixed culture solid state fermentation, pH schematic diagram over time;
Fig. 5 is subtilis HM-66 and plant lactobacillus HM-20 when carrying out mixed culture solid state fermentation, amino-acid nitrogen content schematic diagram over time;
Fig. 6 is after subtilis HM-66 and plant lactobacillus HM-20 carry out mixed culture solid state fermentation 36h, the schematic diagram of the percentage composition of different polypeptide molecular weight.
Embodiment
Below in conjunction with example, the present invention is further described, and following example is illustrative, is not determinate, can not limit protection scope of the present invention with following example.
For plant lactobacillus and the subtilis of fodder additives neck probiotic solid fermentation, subtilis HM-66 and plant lactobacillus HM-20 specific name are respectively BacillussubtilisHM-66 and LactobacillusplantarumHM-20, deposit number is respectively CGMCCNo.6733 and CGMCCNo.6744, preservation date on October 29th, 2012, preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.
For the subtilis of fodder additives probiotic solid fermentation and the application of plant lactobacillus, the cultivation of described subtilis HM-66 and plant lactobacillus HM-20 and solid state fermentation thereof are cultivated, obtains the solid state fermentation soybean products of probiotic bacterium.
For the subtilis of fodder additives probiotic solid fermentation and the using method of plant lactobacillus, comprise the following steps:
1. the seed liquor preparation of thalline
Being received from the glycerine pipe of cold storage by subtilis HM-66 is equipped with in the substratum of liquid seeds, and be positioned on shaking table, rotating speed is 150r/min, and temperature is 37 DEG C, cultivates 20h.Plant lactobacillus HM-20 seed culture condition, is placed in constant incubator quiescent culture, culture temperature 37 DEG C, and incubation time is 20h.Wherein liquid seed culture medium (g/L) is: raffinose 20, dregs of beans 10, pH7.0-7.2.
2. solid state fermentation
Subtilis HM-66 and plant lactobacillus HM-20 is connect bacterium to the wet dregs of beans substratum gone out with 1.0% and 4.0% inoculum size respectively, sealing be placed on constant incubator quiescent culture.
Solid-state fermentation culture medium is: 58% dregs of beans, the sugar of 2%, moisture content 40%, 115 DEG C of autoclaving 25min.
3. measure viable count
General employing colony counting method, operation steps is: take the bean pulp fermentation goods 10g prepared, be placed in the stroke-physiological saline solution of 90ml, be placed on shaking table and shake 30min, shaking up the bacterium liquid that rear liquid-transfering gun gets 1ml adds in the stroke-physiological saline solution of another 9ml, dilute, according to national standard method, take turns doing ten times of gradient dilutions.After having diluted, get suitable extent of dilution 1ml respectively with liquid-transfering gun and throw flat board into, pour into substratum mixing, take turns doing three parallel, be placed in incubator at 37 DEG C of quiescent culture 48h.
The mensuration of 4.pH value
Take the soybean products after 10g fermentation, put into triangular flask, add the distilled water of 90ml.Use magnetic stirrer 30min, after leaving standstill 10min, determine its pH value with Accurate pH measurement.
5. the mensuration of amino-acid nitrogen content
Get the dregs of beans after 10g fermentation, broken with homogenizer, mix with the distilled water of 90ml afterwards, place 1 day at 4 DEG C of refrigerators.The centrifugal 7000r/min of sample, centrifugal 15min.Supernatant liquor 0.8ml is placed in test tube, then adds 3.2ml distilled water, 2.0ml developer, and mixing, puts into boiling water bath and heat 15min, make blank simultaneously.Then cold water cooling, adds the mixing of 5.0ml40% ethanolic soln, uses 1cm cuvette after placing 15min, and returning to zero in 570nm place with blank tube measures A value.
6. dregs of beans peptide molecule flow measurement
Get 10g fermentation after dregs of beans, after homogenizer fragmentation, by gained fermented product by 5 times of stroke-physiological saline solution at 4 DEG C of lixiviate 4h, centrifuging and taking supernatant liquor under 4500r/min condition, powder is got in lyophilize, does not separately inoculate solid state fermentation thing as blank.
Chromatographic condition
Chromatographic column: GESuperdex
tMpeptide10/300GL(10 × 300nm);
Moving phase: 50mM phosphate buffer soln (pH is 7.2, and containing 0.15MNacl);
Post pressure: be less than 18bar;
Flow velocity: 0.5ml/min;
Determined wavelength: 214nm
Column temperature: room temperature
Sample size: 10ul.
7. the packaging of fermented product
There is a large amount of viable bacterias in bean pulp fermentation goods, therefore bean pulp fermentation product must carry out sealing packaging, and in cryopreservation, the quality guaranteed period is approximately 6 months.
Below the determination of some parameters of the production method to the probiotics fermention product taking dregs of beans as matrix, and the optimization of some culture condition.
One, inoculum size is on the impact of viable count
Solid-state fermentation culture medium is: 58% dregs of beans, the sugar of 2%, moisture content 40%, 115 DEG C of autoclaving 25min.
When the bacterium amount that connects of plant lactobacillus HM-20 is 1%, culture temperature 37 ± 1 DEG C, incubation time 28h, recording plant lactobacillus HM-20 viable count is 2.3 × 10
8cfu/g.
When the bacterium amount that connects of plant lactobacillus HM-20 is 4%, culture temperature 37 ± 1 DEG C, incubation time 28h, recording plant lactobacillus HM-20 viable count is 2.8 × 10
8cfu/g.
When the bacterium amount that connects of plant lactobacillus HM-20 is 6%, culture temperature 37 ± 1 DEG C, incubation time 28h, recording plant lactobacillus HM-20 viable count is 1.4 × 10
8cfu/g.
Result: the suitableeest inoculum size of plant lactobacillus HM-20 is respectively 4%(see Fig. 1).
Two, water is soaked on the impact of viable count
When the pH soaking water is 6.0, when the inoculum size of plant lactobacillus HM-20 is respectively 4%, culture temperature 37 ± 1 DEG C, incubation time 32h, recording plant lactobacillus HM-20 viable count is 5.6 × 10
8cfu/g.
When the pH soaking water is 6.5, when the kind amount of plant lactobacillus HM-20 is respectively 4%, culture temperature 37 ± 1 DEG C, incubation time 32h, recording plant lactobacillus HM-20 viable count is 6.9 × 10
8cfu/g.
When the pH soaking water is 7.0, when the kind amount of plant lactobacillus HM-20 is respectively 4%, culture temperature 37 ± 1 DEG C, incubation time 32h, recording plant lactobacillus HM-20 viable count is 3.6 × 10
8cfu/g.
Result: when the pH soaking water is 6.5, the viable count of plant lactobacillus HM-20 is the highest.
Three, subtilis HM-66 inoculum size is on the impact of viable count
When the inoculum size of subtilis HM-66 is 0.6%, when plant lactobacillus HM-20 is inoculum size 4%, soaking water pH is 6.5, and culture temperature 37 ± 1 DEG C, incubation time 32h, recording plant lactobacillus HM-20 viable count is 5.1 × 10
8cfu/g.
When the inoculum size of subtilis HM-66 is 1.0%, when the kind amount of plant lactobacillus HM-20 is 4%, soaking water pH is 6.5, and culture temperature 37 ± 1 DEG C, incubation time 32h, recording plant lactobacillus HM-20 viable count is 6.3 × 10
9cfu/g.
When the inoculum size of subtilis HM-66 is 2.0%, when the kind amount of plant lactobacillus HM-20 is 4%, soaking water pH is 6.5, and culture temperature 37 ± 1 DEG C, incubation time 32h, recording plant lactobacillus HM-20 viable count is 9.5 × 10
8cfu/g.
Result: when subtilis HM-66 and plant lactobacillus HM-20 mixed fungus fermentation, its suitableeest inoculum size is that 1%(is see Fig. 2).
Four, the solid state fermentation time is on the impact of viable count
Bacterial growth experienced by lag phase, logarithmic phase, balance period and paracme respectively, and at different times, growing state is different, carries out live bacterial count respectively to its sampling.
Solid-state fermentation culture medium is: 58% dregs of beans, the sugar of 2%, moisture content 40%, 115 DEG C of autoclaving 25min.
Inoculum size and culture condition: subtilis HM-66 and plant lactobacillus HM-20 inoculum size are respectively 1.0% and 4.0%, soak water pH6.5, culture temperature 37 ± 1 DEG C, incubation time 28h, recording plant lactobacillus HM-20 viable count is 4.2 × 10
8cfu/g.
Subtilis HM-66 and plant lactobacillus HM-20 inoculum size are respectively 1.0% and 4.0%, and soak water pH6.5, culture temperature 37 ± 1 DEG C, incubation time 36h, recording plant lactobacillus HM-20 viable count is 1.9 × 10
10cfu/g.
Subtilis HM-66 and plant lactobacillus HM-20 inoculum size are respectively 1.0% and 4.0%, and soak water pH6.5, culture temperature 37 ± 1 DEG C, incubation time 48h, plant lactobacillus HM-20 viable count is 2.2 × 10
10cfu/g.
Result: when considering production cost and production cycle, as subtilis HM-66 and plant lactobacillus HM-20 mixed fungus fermentation, the suitableeest fermentation time is 36h.
Conclusion
When subtilis HM-66 and plant lactobacillus HM-20 inoculum size are respectively 1.0% and 4.0%, the pH soaking water is 6.5, and culture temperature 37 ± 1 DEG C, after incubation time 36h, its viable count is higher, and the viable count recording plant lactobacillus HM-20 is 1.9 × 10
10cfu/g(is see Fig. 3, Fig. 4, Fig. 5, Fig. 6).
Claims (1)
1. for the mixing thalline of fodder additives probiotic solid fermentation, comprise subtilis (
bacillussubtilis) HM-66 and plant lactobacillus (
lactobacillusplantarum) HM-20, it is characterized in that: described subtilis and plant lactobacillus are probiotic bacteriums, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is respectively CGMCCNo.6733 and CGMCCNo.6744, and preservation date is on October 29th, 2012.
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